O-GlcNAc transferase (OGT), found in all metazoans, is essential for embryonic development in mammals and continues to be required throughout life for the viability of proliferating cells. Although the importance of OGT in biology is not disputed its functions remain poorly understood. It has three distinct biochemical activities: 1) it acts asa glycosyltransferase, attaching N-acetylglucosamine (GlcNAc) to a wide variety of cytoplasmic and nuclear proteins and thereby affecting their stability, localization, and biochemical functions in response to changing cellular conditions; 2) it functions as a protease in the maturation of HCF-1, an essential multi-domain transcriptional co-regulator required for cell cycle progression; and 3) it serves as a scaffolding protein that interacts with components of several multi-protein complexes. OGT has been implicated in diseases involving dysregulated glucose uptake and metabolism, including cancer and diabetes, and it is a proposed therapeutic target. The research proposed here combines chemical synthesis, biochemistry, and cell biology to gain a better understanding of OGT's different activities, which is critical for assessing its potential a a therapeutic target. Aim 1 focuses on the development of cell permeable small molecule inhibitors based on a lead discovered in the previous funding period. These inhibitors will be useful for investigating OGT's cellular activities and are particularly important for studies of it roles in cell signaling. Aim 2 focuses on using protein microarrays to investigate the structural features of OGT that are important for substrate selection. These studies may reveal that different parts of the OGT TPR domain are involved in selecting different subsets of substrates, a result that would have implications for pathway-selective OGT inhibitors. Aim 3 focuses on testing our proposed mechanism for how OGT cleaves HCF-1. These studies are warranted because the mechanism, like the discovery that OGT uses the same active site for both protein O-GlcNAcylation and proteolysis, is unprecedented in biology. Finally, Aim 4 focuses on establishing a genetic system to replace wildtype OGT with OGT variants deficient in a particular biochemical activity so that we can address the following fundamental questions: Why is OGT required for survival of proliferating mammalian cells? Is HCF-1 cleavage required? Is OGT's scaffolding function required? Or is O-GlcNAcylation activity necessary, and, if so, what targets are most important? A robust genetic system to investigate OGT variants will allow us to link findings from biochemical studies to cellular phenotypes, leading to a better understanding of OGT biology.